Abstract 136: Differential Effect of Nitric Oxide on the 26S Proteasome in Type 1 and Type 2 Diabetic Rats
INTRODUCTION: We have shown that nitric oxide (NO) is more effective at inhibiting neointimal hyperplasia in rodent models of type 2 versus type 1 diabetes. To explain this difference, we looked at the 26S proteasome and deubiquitinating enzyme, USP5, since recent data revealed altered protein ubiquitination in diabetic environments. We hypothesize that NO has a differential effect on the 26S proteasome, the proteasomal activator, PA28-α, and/or USP5 in type 1 vs. 2 diabetic rat models.
METHODS: The carotid artery injury model was performed in non-diabetic (LZ), type 1 (streptozotocin-injected LZ [STZ]), and type 2 diabetic (ZDF) rats. Groups included control, injury, and injury+PROLI/NO [n=4-7/group]. At 72hrs, carotid arteries were homogenized. PA28-α and USP5 expression was assessed by Western blot analysis. Vascular smooth muscle cells (VSMC) were harvested from LZ and ZDF rats and exposed to NO. After 24hrs, Western blot analysis for PA28-α and USP5, or the 26S activity assay was performed.
RESULTS: ZDF arteries exhibited 2-fold more PA28-α expression versus STZ arteries. While NO caused a 2-fold decrease in PA28-α after injury in LZ arteries, NO caused complete inhibition in STZ and ZDF arteries versus injury alone. Similarly, ZDF arteries showed 2-fold more USP5 expression versus STZ arteries. While NO caused a decrease in USP5 expression in LZ arteries, NO exposure resulted in a doublet formation and no inhibition of USP5 in STZ and ZDF arteries versus injury alone. NO inhibited 26S proteasome activity (p<0.05), but there were no differences between LZ and ZDF VSMC. NO had no effect on USP5 expression in LZ and ZDF VSMC. However, treatment of VSMC with NO caused a concentration-dependent decrease in LZ and increase in ZDF PA28-α expression.
CONCLUSION: NO caused significant early changes in PA28-α and USP5 in rodent models of type 1 and 2 diabetes versus non-diabetic rats. NO also increased expression of PA28-α in diabetic VSMC, but did not differentially affect proteasomal activity or protein deubiquitination in vitro. These data indicate a strong relationship between protein ubiquitination and degradation and the development of neointimal hyperplasia in diabetic rat models and may lead to a novel mechanism to prevent restenosis in diabetic patients.
- © 2012 by American Heart Association, Inc.